Sutures have been used as a conventional surgical means for uniting tissues and surgical margins, as hemostatic aids, and for blocking or ligation. However, sutures suffer from many drawbacks. For example, sutures may be incompatible with the tissue, causing fistula or granuloma, sutures may cut through parenchymal and inflammatory tissues, absorbable suture material may disintegrate prematurely and produce dehiscence of the wound, and closely spaced sutures may cause tissue ischemia resulting in necrosis of the wound margins. Suturing is also time-consuming.
In order to overcome the above-mentioned shortcomings of sutures, various attempts at developing suitable substitutes have been made. One goal has been the development of a tissue glue which ensures union of the tissue without causing any damage thereto.
Cyanoacrylate-based substances have been commonly used as a glue for tissue. However, these substances are toxic to the tissue and cannot be absorbed. As early as 1909, it was realized that "fibrin powder" could be utilized to achieve blood clotting and wound healing. Due to advances in basic research, it is now possible to prepare highly concentrated plasma products and isolate some coagulation factors.
In the human application, autologous plasma cryoprecipitate solution was first used, but since the clottable substances were found to be insufficient, homologous cryoprecipitate solution from pooled single-donor plasmas was subsequently used to obtain higher concentration for better tensile strength. Later, fibrin glue or sealant became more widely known.
Fibrin sealant has three components: fibrinogen concentrate, calcium chloride and thrombin. These components mimic the final common pathway of the clotting cascade, i.e. the conversion of fibrinogen to fibrin sealant.
In preparing fibrin sealant, thrombin of bovine or human origin is diluted with calcium chloride, with concentrations dependent on the tissue to be applied and the time of clotting. Equal amounts of fibrinogen concentrate and thrombin diluted in calcium chloride are used for clinical application. When the two components are mixed, thrombin converts fibrinogen to fibrin so that clotting is initiated and the mixture solidified. Meanwhile, in the presence of calcium ions, thrombin activates factor XIII to factor XIIIa. Activated factor XIIIa together with thrombin catalyzes the cross-linkage of fibrin and increases the strength of the clot. During wound healing the clot material undergoes gradual lysis and is completely absorbed. A major application of fibrin sealant is in surgery and in other areas of medicine.
Although there are great advantages to using fibrin sealant in clinical medicine, it is prohibited to use the commercially available product from pooled human plasma in the United States because of potential transmission of hepatitis B, acquired immunodeficiency syndrome (AIDS), and other transfusion transmittable diseases. The Food and Drug Administration (FDA) regulations have required that all plasma protein fractions shall receive heat treatment for not less than 10 or more than 11 hours at an attained temperature of 60.degree..+-.0.5.degree. C. to inactivate infectious agents.
Commercially available fibrinogen is prepared from the plasma pooling of a large number of donors, which has high potential for disease transmission. In addition, fibrinogen will not tolerate the ten hours of heating to 60.degree. C. used to inactivate the hepatitis virus in other blood fractions. Studies have indicated that this product was a source of hepatitis transmission (7.8% of post-transfusion hepatitis rate). Under these circumstances, the FDA revoked all licenses for the manufacture of human fibrinogen since Jun. 30, 1978.
In Europe, fibrinogen product is commercially available as a fibrinogen concentrate kit ("Tisseel", Immonu AG, Vienna, Austria) prepared from pooled fresh frozen plasma. The tensile strength for Tisseel is 900 /g/cm.sup.2. Since this commercial fibrinogen concentrate is not available in the United States because it is currently not licensed by the FDA, alternative methods such as chemical precipitation and cryoprecipitation have been used to prepare fibrinogen concentrate.
Fibrinogen is one of the three main protein constituents of plasma. The major constituent, albumin (ALB), occurs in a concentration of approximately four percent. The plasma globulins are present in a concentration of about 2.5 percent and are particularly associated with-the processes of immunity. Fibrinogen occurs in much smaller amounts, with its concentration in human plasma being about 0.4 percent.
Several authors have discussed fibrinogen/fibrinogen interaction and fibrinogen interaction with other proteins. Aggregation of fibrinogen at pH 5.7 and low ionic strength (&lt;0.3) has been found. A disulfide bond between fibrinogen molecules in cold-insoluble fibrinogen fraction has been demonstrated. It has been thought that the cold-insoluble precipitate that formed from normal plasma was a reaction between cold-insoluble globulin (CIg), fibrinogen and fibrin.
The plasma proteins can be separately isolated by: 1) organic solvents such as methanol or ethanol at low temperature using Cohn's fractionation, 2) cryoprecipitation, 3) chemical precipitation of plasma with salts such as ammonium sulfate, potassium phosphate, and sodium citrate, and 4) other methods. The solubility of the plasma proteins in these substances decreases in the order of albumin, globulin, and fibrinogen. The latter precipitates first and albumin last upon the addition of increasing amount of the precipitating agent.
1. Ethanol Fractionation (Cohn's fractionation)
In this process, 1,000 to 1,500 liters of 4,000-6,000 human source plasma are pooled and treated sequentially in the cold with various concentrations of ethanol and buffers to precipitate fractions containing different plasma proteins. Fibrinogen is the first material precipitated and harvested at -5.degree. C. with 25% ethanol at a pH of 6.9. Variables determining the precipitation of proteins are ethanol concentration, pH, temperature, ionic strength and protein concentration.
2. Cryoprecipitation
The standard cryoprecipitation method has been primarily used to prepare antihemophilic factor (Factor VIII). Cryoprecipitate also has been known as a source of fibrinogen. The cryoprecipitate method can be also used to prepare fibrinogen concentrate. It is known that some factors might affect the yield of Factor VIII, such as ABO blood grouping, freezing and thawing conditions. With respect to Factor VIII preparation, others have studied freezing and thawing conditions (see Brown, et al., "Antihaemophilic Globulin: Preparation by an Improved Cryoprecipitation Method and Clinical Use", Br Med J 2, 79-85, 1967). However, all the factors for cryoprecipitation are not known.
It has been observed that when frozen plasma is thawed in the cold at 4.degree. C., most of the Factor VIII remains in the cold-insoluble precipitate. This precipitate also contains variable amounts of fibrinogen ranging from 100 to 300 mg/single donor unit of cryoprecipitate. It has become routine to prepare anti-hemophilic factor (Factor VIII) and fibrinogen using the cryoprecipitation method in the blood bank using a closed system of plastic bags to maintain the sterility of the product from collection of the whole blood from the donor.
3. Chemical Precipitation
Human fibrinogen can be precipitated from human plasma by ammonium sulfate, polyethylene glycol, plyvinyl-pyrrolidone, and barium/magnesium sulfate. Entering the closed blood bag system for the addition of chemicals opens the system to the potential for bacterial contamination. Small amounts of fibrinogen concentrate solution (0.5-1.9 ml) can be prepared using these methods, but the side effects and safety due to the chemical substances as well as bacterial contamination opportunities are of great concern.
4. Other Methods
Sporadic reports have mentioned the use of the following methods to prepare purified fibrinogen: chromatography, polyelectrolyte fraction technology, recombinant DNA technology and ion exchange chromatography.
Fibrinogen concentrate can be prepared from random single-donor fresh frozen plasma or autologous plasma in sufficient quantity to meet some surgical demand. According to the Standards of the American Association of Blood Banks, fibrinogen concentrate can be currently stored for up to 5 years at -80.degree. C. or at least 5 days at 4.degree. C. until it is needed. Cryoprecipitate contains Factor VIII and fibrinogen and is used to supply fibrinogen in patients with hypofibrinogemia and also as an alternative source of fibrinogen concentrate for fibrin sealant in the United States.
However, traditional cryoprecipitation suffers from problems including the recovery of only small amounts of fibrinogen having low tensile strength when using single-donor cryoprecipitate to prepare fibrin sealant. Further, the fibrinogen concentrates prepared by traditional cryoprecipitation have a concentration range of 260-2,500 mg/dl. This is not an adequate concentration for applying this product as a tissue sealant over highly vascular areas. High fibrinolytic activity over that area breaks down the fibrin clot very quickly. These concentrates have a tensile strength of around 120 gm/cm.sup.2 which is usually not sufficient for surgical applications.
The following patents reflect the state of the art of which applicant is aware insofar as these patents appear germane to the patent process. However, it is respectfully stipulated that none of these patents teach singly nor render obvious when considered in any conceivable combination the nexus of the instant invention as set forth hereinafter.
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